Evolved resistance to a novel cationic peptide antibiotic requires high mutation supply.

Background and Objectives: A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Methodology: Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results: Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusions and implications: The results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.

Characterization of Female Reproductive Proteases in a Butterfly from Functional and Evolutionary Perspectives.

Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae, the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.